SUMMARY The syndrome of delayed anaphylaxis to red meat, i.e. the alpha-gal syndrome (AS), is found exclusively in patients who have serum IgE antibody (ab) to the oligosaccharide galactose-alpha-1,3-galactose (alpha-gal). This form of sensitization is related to bites of the tick Amblyomma americanum, however other ticks appear to be relevant in other countries. The objective of the studies proposed here is to investigate the epidemiology, etiology, immunology and clinical features of AS which is now known to be common in a large area of the USA. The studies focus on three aspects of the syndrome. Firstly we will use a detailed map of the cases of AS to compare with maps of tick-related diseases and the distribution of ticks. The serologic response to alpha-gal will be compared from AS cases in participating sites from different parts of the country. Additionally, to investigate the possibility that the Rickettsiae group of obligate intracellular bacteria, such as Rickettsia amblyommii or Ehrlichia chaffeensis, could be relevant to AS sensitization we will carry out assays for specific IgG ab. Secondly, in the area where there is a high prevalence of IgE to alpha-gal there are many individuals who are sensitized but either do not report symptoms or report delayed symptoms that are not recognized as allergic. These include patients with repeated episodes of abdominal pain. Our primary hypothesis about the 3- 5 hour delay in the onset of symptoms is that this reflects the time taken to process dietary mammalian glycoprotein and/or glycolipids lipids to a size that readily penetrates into tissue, for example the size of LDL (~20 nm). Another disease not traditionally considered to be related to allergy is coronary artery disease. In a preliminary investigation we have found that the presence of detectable levels of IgE to alpha-gal is significantly associated with the burden of atherosclerosis in a cohort of subjects in central Virginia. This study, which utilized intravascular ultrasound (IVUS), also demonstrated an association with coronary plaques which had unstable, i.e. high risk, features. We will further investigate this association in a larger cohort at our institution and extend the investigation to subjects in other regions with high prevalence of alpha-gal sensitization. The primary objective of the third aim is to obtain better information about the B cells that relate to the antibody response to alpha-gal. Preliminary data using mass cytometry suggests that subjects with AS have a population of plasmablasts that can respond to alpha-gal. The proposed studies will further investigate these cells with an expanded mass cytometry bead panel and use multiple samples in a single batch. In conjunction with parallel studies employing antibody deep sequencing, collective results are expected to shed insight into the B cell populations that produce `natural' antibodies to alpha-gal, i.e. IgM and IgG2 antibody, as well as those B cells that produce IgE and IgG1. Finally, we will compare the response in AS with that in eosinophilic esophagitis (EoE) which is another form of food allergy with a delayed response and a dramatically different antigen-specific IgE and IgG4 response.